Nucleotide vector, composition containing such vector, and vaccine for immunization against hepatitis

ABSTRACT

Nucleotide vector composition containing such vector and vaccine for immunization against hepatitis. Nucleotide vector comprising at least one gene or one complementary DNA coding for at least a portion of a virus, and a promoter providing for the expression of such gene in muscle cells. The gene may be the S gene of the hepatitis B virus. A nucleotide vector composition when administered to even chronic HBV carriers is capable of breaking T cell tolerance to the surface antigens of hepatitis B virus. A vaccine preparation containing said bare DNA is injected into the host previously treated with a substance capable of inducing a coagulating necrosis of the muscle fibers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/706,337, filed Aug. 30, 1996, which is a continuation-in-part ofapplication Ser. No. 08/633,821, filed Apr. 22, 1996, which is based onInternational Application PCT/FR94/00483, filed Apr. 27, 1994. Theentire disclosure of each of these applications is relied upon andincorporated by reference herein.

BACKGROUND OF THE INVENTION

The present invention relates to compositions for inducing protectiveantibodies against hepatitis. It also relates to a vector comprising anucleotide sequence coding for at least a portion of a virus protein,which is capable of being expressed in muscle cells. In addition, theinvention relates to compositions capable of inducing a T cell responsein chronic HBV carriers.

Hepatitis B is a widespread and serious international health problem. Inaddition to causing acute hepatitis and liver damage, the hepatitis Bvirus (HBV) can cause cirrhosis and hepatocellular carcinoma (Davis,Hum. Molec. Genet. 2:1847-1851 (1993)).

The HBV is a 42-nm particle (Dane particle) consisting of a lipoproteinenvelope enclosing a core protein (capsid) and the viral genome, whichcontains only four genes (S, C, P, X). The major (or small) envelopeprotein, which includes the surface antigen of HBV (HBsAG) is encoded bythe S gene and is organized into dimers of one glycosylated and oneunglycosylated polypeptide (Petersen, J. Biol. Chem. 256:6975-6983(1981)). Present in smaller amounts are the middle and large envelopeproteins, which are encoded by the pre-S2 and S or pre-S1, pre-S2 and Sgenes, respectively. The predominant form of HBsAg secreted by infectedcells is not the Dane particle, however, but 22-nm particles orfilaments, which are empty viral envelopes composed solely orpredominantly of major (small) envelope protein and sometimes smallamounts of middle and large proteins (Maupas, Lancet 1:1367-1370(1976)). The 22-nm particles are seen to persist in the plasma ofchronic carriers (Davis, 1993).

In 1976, the first vaccine against HBV comprising 22-nm HBsAg particleswas applied to humans (Maupas, 1976). The particles were purified fromthe plasma of chronic carriers and treated to eliminate possibleco-purified infectious HBV or other pathogens. While this vaccine waseffective, mass immunization was not feasible due to the long andexpensive purification procedure, the need to assay each batch onchimpanzees for safety, and the limited supply of chronically infectedhuman plasma (Maugh, Science 210:760-762 (1980); Stephenne, Vaccine6:299-303 (1988)).

The present vaccines are produced employing genetic engineeringtechniques to create HBsAg-producing cell lines. One frequently usedvaccine is a second generation vaccine based on recombinant yeast cellscontaining the S gene for HBsAg (Valenzuela, Nature 298:347-350 (1982)).Another vaccine commonly used in France is a third generation vaccinebased on a line of Chinese hamster ovary cells containing both the S andpre-S2 genes (Michel, Proc. Natl. Acad. Sci. USA 81:7708-7712 (1984)).While the present protein vaccines are highly effective and safe, theproduction and maintenance of these vaccines is time-consuming andexpensive (Davis, 1993). On the other hand, the production of a viralvaccine is not feasible due to safety considerations.

Immunization by DNA-based vaccines has been the object of severalstudies since the beginning of the 1990s. A DNA-based vaccine involvesthe transfer of a gene or at least a portion of a gene, by direct orindirect means, such that the protein subsequently produced acts as anantigen and induces a humoral- and/or cellular-mediated immunologicalresponse.

Ulmer et al. (Science, 259:1745-1749 (1993)) obtained protection againstthe influenza virus by induction of the cytotoxic T lymphocytes throughinjection of a plasmid coding for the influenza A nucleoprotein into thequadriceps of mice. The plasmid used carries either the Rous sarcomavirus promoter or the cytomegalo virus promoter.

Raz et al. (Proc. Natl. Acad. Sci. USA 90:4523-4527, (1993)) injectedvectors comprising the Rous sarcoma virus promoter and a gene coding forinterleukin-2, interleukin-4, or the β1-type transforming growth factor(TGF-β1). The humoral and cell-mediated immune responses of the mice towhich these plasmids have been intramuscularly administered areimproved.

Wang et al. (Proc. Natl. Acad. Sci. USA, 90:4156-4160, (1993)) injecteda plasmid carrying a gene coding for the envelope protein of the HIV-1virus into mice muscles. The plasmid injection was preceded by treatmentwith bupivacaine in the same area of the muscle. The authorsdemonstrated the presence of antibodies capable of neutralizing theHIV-1 virus infection. However, the DNA was injected twice a week for atotal of four injections.

Davis et al. (Compte-Rendu du 28 ème Congres Européen sur le muscle,Bielefeld, Germany, 21-25 Sep. 1992) injected plasmids carrying aluciferase or β-galactosidase gene by pretreating the muscles withsucrose or a cardiotoxin. The authors observed the expression ofluciferase or β-galactosidase.

More recently, an article published in Science et Avenir (September1993, pages 22-25) indicates that Whalen and Davis succeeded inimmunizing mice against the hepatitis B virus by injecting pure DNA fromthe virus into their muscles. An initial injection of snake venom toxin,followed 5 to 10 days later by a DNA injection, is generally described.However, the authors specify that this method is not practical.

These studies were preceded by other experiments in which various DNAswere injected, in particular into muscle tissues. For example, theInternational application, PCT/US90/01515 (published under No. WO-90/11092), discloses various plasmid constructions, which can be injected inparticular into muscle tissues for the treatment of muscular dystrophy.However, this document specifies that DNA is preferentially injected inliposomes.

Additionally, Canadian patent CA-362.966 30 (published under No.1,169,793) discloses the intramuscular injection of liposomes containingDNA coding, in particular, for HBs and HBc antigens. The resultsdescribed in this patent mention the HBs antigen expression. Thepresence of anti-HBs antibodies was not investigated.

International application PCT/FR92/00898 (published under No.WO-93/06223) discloses viral vectors, which can be conveyed to targetcells by blood. These vectors are recognized by the cell receptors, suchas the muscle cells, and can be used in the treatment of musculardystrophy or of thrombosis.

The DNA-based vaccines suggested by the prior art have not been capableof practical uses. For example, some bare DNA used to vaccinate the micewas pure DNA from the virus. This type of treatment can not beconsidered for human vaccination due to the safety risks involved forthe patients. Additionally, earlier experiments in which the injectedDNA is contained in liposomes did not exhibit an immune response.

The present inventors have succeeded in developing effective DNA-basedimmunizing compositions capable of inducing immune responses againstinfectious viruses without the detrimental effects on human health.

SUMMARY OF THE INVENTION

The present invention relates to a composition capable of inducing Tcell response, and more particularly, a cytotoxic response comprising anucleotide sequence expressed in muscle cells. The nucleotide sequencecomprises a gene or complementary DNA coding for at least a portion of avirus protein and a promoter allowing for the expression of the gene orcomplementary DNA in the muscle cells.

The invention further relates to the vector, which serves as a vehiclefor the gene or complementary DNA coding for at least a portion of avirus protein and a promoter allowing for the expression of the gene orcDNA, which is administered to an individual to be immunized.

In addition, the inventors have developed a non-lipid pharmaceuticalcomposition comprising at least, on the one hand, a substance capable ofinducing a coagulating necrosis of the muscle fibers, such asbupivacaine, and, on the other hand, vector including the gene orcomplementary DNA coding for at least a portion of a virus protein,which is expressed in muscle cells, and the promoter. Preferably, thesubstance capable of inducing a coagulating necrosis of the musclefibers is first administered into the muscle of an individual to beimmunized. Then, at least five days later, the vector is administeredinto substantially the same location of the individual's muscle.

The inventors discovered that the compositions of the instant inventionare capable of breaking T-cell tolerance to HBsAg in a mouse model forchronic HBV carriers. Thus, the present invention is further directed tothe treatment of chronic HBV carriers.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be described in greater detail with reference to thedrawings in which:

FIG. 1 is a schematic representation of pRC/CMV-HBs plasmid.

FIGS. 2A to 2D are schematic representations of pCMVHB-S, pCMVHB-S2.S.,pCMVHB-S1.S2.S and pHBV-S1.S2.S plasmids, respectively.

FIGS. 3, 4, and 5 are schematic restriction maps for pCMVHB-S2.S,pCMVHB-S1.S2.S, and pRSV-HBS plasmids, respectively:

FIG. 6 illustrates the secretion of antigenic HBs particles (HBs Ag) inng/ml (ordinates) as a function of the number of days (abscissa) forcells carrying the pCMVHB-S, pCMVHB-S1.S2.S, pHBV-S1.S2.S, pSVS, orpCMVHB-S2.S plasmids.

FIGS. 7A and 7B illustrate the determination on some particles in FIG. 6of the presence of the preS₁ and PreS₂ antigens using, respectively,anti-preS₁ and anti-preS₂ antibodies. The formation of antibody-antigencomplexes is shown by the optical density (ordinates), as a function ofantigen concentration.

FIGS. 8A to 8D represent the anti-HBS responses (HBS Ab as ordinate,expressed as mUI/ml) and anti-preS2 (preS2 Ab as ordinate, expressed inO.D.) of mice vaccinated by pCMVHB-S (8A), pCMVHB-S2.S (8B),pCMVHB-S1.S2.S (8C), and pHBV-S1.S2.S (8D), respectively.

FIG. 8E depicts the kinetics of appearance of anti-pre-S2 andanti-pre-S1 antibodies in sera from groups of mice injected with HBVenvelope-expressing plasmids (pSVS and pCMVS1S2S).

FIGS. 8F and 8G illustrate the kinetics of IgG and IgM anti-HBs in miceimmunized with HBV envelope-expressing plasmids. The fine specificity ofthe antibodies was determined using S-containing HBsAg of a heterologoussubtype (ad; circles) of a homologous subtype (ay; triangles), as wellas HBsAg containing approximately 30% of the middle (pre-S2 and S)protein of the ay subtype (squares). The bound anti-mouse IgG antibodiesare depicted as a continuous line and bound anti-mouse IgM antibodiesare depicted as a dotted line.

FIG. 8H depicts the anti-HBs immune response in mice injected with thetwo expression vectors pSVS (squares) and pCMV-S1.S2.S (circles).

FIG. 9 illustrates the antibody response, IgG and IgM immunoglobulins(titre as ordinates), of a mouse vaccinated by pCMVHB-S2.S as a functionof the number of weeks (abscissa).

FIGS. 10A to 10C represent the anti-group and anti-subtype ay responsesinduced by DNA from pCMV-S (DNA) or from the HBS antigen (prot),respectively, in mice B10 (10A), B10S (10B), and B10M (10C).

FIGS. 10D to 10F represent the antigroup responses induced by DNA frompCMV-S (DNA) or from the HBS antigens (prot), respectively in mice B10(10D), B10S (10E), and B10M (10F).

FIG. 11 represents a linear restriction map for the pBS-SKT-S1.S2.Splasmid.

FIG. 12 represents the DNA-based immunization of transgenic mice. Groupsof 6 female transgenic mice were immunized once by intramuscularinjection of 100 μg of the DNA plasmid pCMV-S2.S (--) or pCMV-LacZ(-□-) five days after cardiotoxin treatment. A group of 8 transgenicmice were injected with PBS instead of DNA (non-immunized controls(−Δ−). Mice were bled at weekly intervals and the sera were analyzed forHBsAg (expressed as ng/ml). Each point represents the mean titre for thegroup and error bars represent the standard errors of the mean.

FIG. 13 describes the kinetics of appearance of anti-HBs antibodies inmice following injection of pCMV-S2.S DNA. Sera were taken as in FIG. 12and the fine specificity of the antibodies was determined using HBsAgparticles containing either the S (open symbols) or the S plus preS2proteins (filled symbols). Anti-HBs antibodies (Ig) were expressed as1/log₁₀ of the antibody titre (determined by serial end-point dilutionanalysis). Circles represent the immunized transgenic mice shown in FIG.1, diamonds are non-transgenic immunized mice, and squares aretransgenic mice injected with pCMV-LacZ. Symbols in the grey areacorrespond to mice which gave no detectable seroconversion (titre <100).

FIG. 14 provides an anti-HBs IgG isotype profile in the sera of sixindividual transgenic mice (solid columns) and three non-transgenic mice(open columns) at 12 weeks after immunization with pCMV-S2.S DNA.HBsAg-specific IgG1, IgG2a, IgG2b, and IgG3 antibodies were detected byELISA with specific secondary antibodies. Antibody titres are expressedas a serial end-point dilutions, which was defined as the highest serumdilution that resulted in an absorbance value two times greater thanthat of non-immune or control serum, with a cutoff value of 0.05.

FIG. 15 describes the expression of HBV sequences in the livers oftransgenic and non-transgenic mice. Northern blot analysis of 50 μg oftotal RNA isolated from the livers of transgenic mice (+) and theirnon-transgenic littermates (−) after direct injection of DNA (A) or 26days after adoptive transfer of primed spleen cells (B). ³²P-labelledDNA probes specific for HBV and β-actin were used. The molecular weights(Kb) of the two mRNAs encoded by the transgene are indicated.

A. Lane 1: non-immunized transgenic mouse.

Lanes 2-4: pCMV-LacZ immunized transgenic mice.

Lanes 5-9: transgenic mice immunized with pCMV-S2.S DNA.

B. Transgenic mice receiving primed spleen cells harvested fromnon-transgenic mice 3-6 weeks after immunization with pCMV-LacZ (lanes2-3) or with pCMV-S2.S (lanes 4-8). Lane 9: transgenic mouse receivingunprimed spleen T cells. Lane 1: RNA from the liver of non-transgenicmouse is shown as a negative control. The transferred spleen cellpopulation is indicated on the top. HBsAg titres (ng/ml) and the present(++) or the absence (−) of HBs antibody at the time of sacrifice areindicated.

FIG. 16 describes adoptive transfer of primed spleen cells intotransgenic mice. Non-transgenic mice were immunized by intramuscularinjection of pCMV-S2.S or pCMV-LacZ DNA in order to produce primedspleen cells for adoptive transfer into their transgenic littermates.The mean titre of antibodies in the serum of donor mice at the time ofspleen harvest was 1×10⁵. Eleven pCMV-S2.S recipients were bled at 2 or3 days intervals and their sera were analyzed for HBsAg (ng/ml) (-▪-)and antibodies to HBsAg (-□-), (ELISA, end-point dilution titres).Results are shown as mean titres +/−SEM.

(--): Mean titres of serum HBsAg in five control transgenic recipientmice receiving either unprimed pCMV-LacZ-primed spleen cells.

FIG. 17 describes the HBV mRNA content in the livers of transgenic micetaken at various times after adoptive transfer of HBsAg primed spleencells. Northern blots were performed as in FIG. 15 and the quantitativedetermination of the HBV mRNA was done by phosphoimager analysis aftercorrection for mRNA loading and variations in transfer efficiency asassessed by β-actin expression. The results are expressed as arbitraryunits and represented by grey columns. Background level of hybridizationis shown for RNA extracted from a non-transgenic mouse liver (opencolumn on right). Serum-HBsAg concentrations (ng/ml) at the time ofliver harvest are shown (-∘-).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention concerns a composition capable of inducing acytotoxic response against viruses, such as hepatitis B. The compositioncomprises a nucleotide sequence comprising a gene or complementary DNAcoding for at least a portion of a virus protein, wherein said gene orcomplementary DNA is capable of being expressed in muscle cells, and apromoter. The gene or complementary DNA and the promoter is preferablycarried by a vector for administration into an individual to beimmunized or treated for infection. The composition is furtherpreferably capable of inducing protective antibodies.

The nucleotide sequence of a gene or complementary DNA may code for atleast a portion of a viral protein. Said “at least a portion” of a viralprotein signifies, in the context of the invention, an antigenic portionof a protein with which to induce a humoral or cell-mediated immunogenicresponse, such as a cytotoxic response.

Moreover, the protein can be either a structure protein or a regulatoryprotein. Preferably, the protein is a structure protein.

In addition, the viral protein may be derived from any infectious virusagainst which a humoral or cell-mediated immunogenic response isdesired. For example, the virus may be a hepatitis virus, such ashepatitis A, hepatitis B, or a non-A, non-B hepatitis virus, such ashepatitis C, E, or delta. Alternatively, the virus may be anon-hepatitis virus, such as HIV-1.

In a preferred embodiment of the invention, the gene or complementaryDNA codes for at least a portion of the surface antigen of hepatitis B(HBsAg), particularly, in the S, preS2-S, or preS1-preS2-S form ofHBsAg, and where the gene encodes envelope proteins.

Alternatively, the gene made code for HBsAg/ayw.

The gene or protein sequences for hepatitis A, hepatitis B, and non-A,non-B hepatitis viruses, such as hepatitis C, E, or delta, are describedby the following documents, which are relied upon and incorporated byreference herein:

French Patent FR 79 21 811;

French Patent FR 80 09 039;

European Patent EP 81 400 634;

French Patent FR 84 03 564;

European Patent EP 91 830 479; and

Najarian et al., Proc. Natl. Acad. Sci. USA, 82:2627-2631 (1985).

Alternatively, the gene or complementary DNA may code for at least aportion of the gp160 protein of HIV-1 virus associated with the p25protein and/or the p55 protein and/or the p18 protein and/or the Revprotein of HIV-1 virus.

In yet another alternative embodiment, the gene or complementary DNAcodes for a protein from a pathogenic microorganism such as thebacterium causing diphtheria, whooping cough, listeriosis, the tetanustoxin, etc.

The promoter is selected for its ability to allow the efficientexpression of the gene or complementary DNA in the muscle cells. It maybe heterologous, not naturally found in the host, or preferably,homologous, while being originally active in a tissue other than themuscle tissue. The promoter may be an internal or endogenic promoter,i.e., a promoter of the virus from which the gene or cDNA is taken. Sucha promoter may be completed by a regulatory element of the muscle oranother tissue, in particular, an activating element. Alternatively, thepromoter may be from a gene of a cytoskeleton protein, such as thatdescribed by Bolmon (J. Submicros. Cytol. and Patholog., 22:117-122(1990)) and Zehnlin (Gene 78:243-254 (1989)). The promoter mayalternatively be the promoter from the virus HBV surface genes.

In a preferred embodiment, the promoter is advantageously the promoterfor cytomegalovirus (CMV).

The vector of the present invention comprises nucleotide sequence asdescribed above. In particular, the vector comprises the DNA orcomplementary DNA coding for at least a portion of a virus as definedabove and a promoter allowing the expression of the nucleotide sequencein muscle cells.

The vector must be capable of gene transfer of the nucleotide sequenceinto the muscle cells. In addition, the vector is selected in order toavoid its integration into the cell's DNA, since such integrations areknown to activate the oncogens and induce cell canceration. Thus, thevector may be non-replicative.

In an alternative embodiment, the vector may be replicative, which wouldallow a high number of copies per cell to be obtained and the immuneresponse to be enhanced.

Suitable vectors include but are not limited to plasmids, adenoviralvectors, retroviral vectors, and shuttle vectors. Plasmids are thepreferred vector according to the invention.

In a further preferred embodiment, the plasmid is partly bacterial inorigin and notably carries a bacterial replication origin. Furtherpreferred is a plasmid carrying a gene allowing for its selection, as isknown in the art, such as a gene for resistance to an antibiotic.

The vector may also be provided with a replication origin allowing it toreplicate in the muscle cells of its host, as is known in the art, suchas the replication origin of the bovine papilloma virus.

In addition, the vector may include a terminal transcription sequencesituated downstream of the gene.

The vectors may be obtained by methods known by those having ordinaryskill in the art. For example, methods of obtaining these vectorsinclude those by synthesis or by genetic engineering methods. Suchmethods are described, for example, in the technical manual Maniatis T.et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbour: NewYork (1982).

Other suitable vectors are described. The pCMV/HBS or pRCCMV-HBSplasmid, having the SEQ ID No. 1 sequence, was filed under No. I-1370with the Collection Nationale des Cultures des Micro-organismes del'Institut Pasteur (CNCM) on 21 Oct. 1993.

Additionally, the pRSV/HBS plasmid filed under. No. I-1371 with the CNCMon 21 Oct. 1993 is suitable for the instant invention. This plasmid hasa similar structure to pCMV/HBS, but includes the Rous sarcoma virus(RSV) promoter instead of the cytomegalovirus (CMV) promoter.

Other plasmids may be:

-   -   pCMVHB-S1.S2.S constructed by inserting the fragment BglII-BglII        of the S gene, obtained from pCP10, into a pBlueScript vector        modified to contain supplementary cloning sites in the        “polylinker” portion. The fragment containing the S gene was        then removed by KpnI-BssHII digestion then cloned into the        corresponding sites of pcDNA 3 (In vitrogen, Rad Systems Europe        Ltd, Abingdon UK) so as to obtain pCMVHB-S1.S2.S. This plasmid        was filed under No. I-1411 with the CNCM;    -   pCMVHB-S2.S was obtained by eliminating the pre-S1 part of the        HBs gene from pCMVHB-S1.S2.S by KpnI/MstII digestion, then by        bonding the two extremities after treatment with 51 nuclease.        pCMVHB-S2.S was filed with the CNCM under No. I-1410;    -   pHBV-S1.S2.S, filed with the CNCM under No. I-1409, was obtained        by inserting the S gene BglII-BglII fragment, obtained from        pCP10, into a pBlueScript vector modified to contain        supplementary cloning sites in the “polylinker” portion;    -   pBS-SKT-S1.S2.S codes for the three envelope proteins S, S-preS₁        and S-preS₁-preS₂ of the HBV virus; and    -   pSVS codes for the three envelope proteins, S, preS₂-S, and        preS₁-preS₂-S of the HBV virus. The construction of the pSVS        plasmid is described in EP 0 156 712 B1, which is incorporated        herein by reference. Moreover, pSVS has been deposited in the        C.N.C.M. under No. I-1840 on Jan. 30, 1997.

The plasmid DNA may be administered in naked form or in a liposomeformulation.

The present invention further relates to nucleotide sequences comprisinga promoter homologous to the host and another regulatory sequence forthe expression of a gene or complementary DNA coding for one of theabove-mentioned proteins.

The invention is also directed to a vaccine or medicine containing atleast one vector, or a nucleotide sequence, such as defined above. Thevaccine or medicine according to the invention is capable ofinducing-protective antibodies against viruses such as hepatitis B.

Alternatively, the vaccine or medicine according to the invention iscapable of inducing a T cell response in chronic HBV carriers. Thus, theinvention relates to the treatment of chronic HBV carriers, wherein acomposition of the invention is administered into the muscle of thecarrier in a therapeutically effective amount.

“Chronic HBV carriers” are defined as individuals, particularly mammals,initially infected by HBV, who fail to resolve their infection. Theseindividuals may remain persistently infected by HBV and do not appear tobe capable of eliciting a multispecific polyclonal immune response toseveral HBV antigens, as compared to those individuals capable ofclearing the virus following acute infection. In actively infectedchronic patients, the virus replicates in the liver and the disease ismostly mediated by the immune response. Such viral replication is notdetected in other individuals.

Large amounts of empty viral particles carrying the HBsAg are producedand secreted by the hepatocytes of chronic HBV carriers. These particlespersist in the serum and the corresponding HBsAg-specific antibodies(anti-HBs) are not induced or remain undetectable by the conventionaltechniques due to the presence of immune complexes. Thus, tolerance toHBsAg is characteristic of chronic HBV carriers.

The present invention relates to breaking the tolerance to HBsAg inorder to control the infection. The inventors discovered that the lackof T cell response in a mouse model of chronic HBV carriers may beresponsible for this tolerance. Thus, the “treatment” of said chronicHBV carriers relates to the inducement of an immune response to breakthe T-cell tolerance to HBsAg and a “therapeutically effective amount”is said to be that amount, which induces the immune response to breakthe T-cell tolerance.

In a preferred embodiment, the composition administered to chronic HBVcarriers comprises a plasmid carrying the S2.S form of HBsAg andparticularly, is pCMV-S2.S.

The present invention further relates to a composition capable ofinducing a cytotoxic response comprised of at least one nucleotidesequence expressed in the muscle cells and a promoter such as definedabove.

The present invention further relates to a non-lipid pharmaceuticalcomposition for immunizing an individual against a viral infection, suchas a hepatitis, including, on the one hand, a substance capable ofinducing a coagulating necrosis of the muscle fibers, such asbupivacaine, and, on the other hand, vector such as described above orincluding one of the nucleotide sequences coding for at least a portionof a virus protein, complete or partial, capable of being expressed inmuscle cells. The “partial sequence” is a sequence coding for at leastsix (6) amino acids.

The substance capable of inducing a coagulating necrosis of the musclefibers is preferably bupivacaine.

The substance capable of inducing a coagulating necrosis of the musclefibers is administered into the muscle of an individual to be immunized,followed at least five (5) days later by administration of the vectorinto the muscle of an individual to be immunized. Preferably, theadministration of the substance and the vector is substantially in thesame location in the individual's muscle. In another preferredembodiment, the vector is administered ten (10) days afteradministration of bupivacaine and substantially in the same location ofthe individual's muscle.

The prior administration of bupivacaine has demonstrated an unexpectedincrease in the effectiveness of the vector administration as well as inthe immunization of the individual. Thus, the invention further relatesto a method of increasing the effectiveness of DNA-based vaccines, suchas those described above.

The compositions of the present invention may contain additives, whichare compatible and pharmaceutically acceptable.

Moreover, the compositions of the present invention may be administeredby means known in the art and preferentially by intramuscular orintradermal injection. The injection can be carried out using a syringedesigned for such use or a liquid jet gun as described by Furth (Anal.Biochem., 205:365-368 (1992)).

The effective amount of bupivacaine used is that which obtainssufficient degeneration of the muscle tissue in order to achieve optimalimmunization. An injection dosage of about 0.1 mg to about 10 mg perinjected composition is usually suitable.

The effective amount of the vector to be injected is that amounteffective to achieve optimal immunization or immunotherapeutic treatmentof the individual against the virus of interest according to the proteincoded by the gene carried by the vector. An injection dosage of about0.1 to about 1000 μg of vectors per individual is usually suitable.

The present invention is illustrated by, without in any way beinglimited to, the following examples.

Example 1 Induction of Antibodies Against a Hepatitis B Surface Antigenby Sequential Injection of Bupivacaine and of a Plasmid Carrying a GeneCoding for the Antigen

(1) Materials and Methods

1.1 Bupivacaine Pretreatment

All experiments were made on the muscles of the anterior tibia (AT) ofmice C57BL/6 aged between 5 to 7 weeks.

A single degeneration-regeneration cycle of the muscle fibers is inducedin the muscles of the anterior tibia of non-anaesthetized mice, byintramuscular injection of 50 μl marcaine (bupivacaine 0.5%, DMSO 1%)sold by Laboratoires Astra, France. The solution is injected using atuberculosis syringe with a needle fitted into a polyethylene sleeve inorder to limit the penetration depth to 2 mm.

As marcaine is an anesthetic, injections into the right and left legswere performed at 10 to 30 minute intervals to prevent an overdose.

1.2 DNA Preparation

The plasmid used was constructed by cloning into a modified pBlueScriptvector of the XhoI-BglII restriction fragment of the pCP10 plasmid,which contains the gene coding for the HBs surface antigen and thenon-translated sequences, both upstream and downstream, including thepolyadenylation signal.

The S gene was then recovered by digestion using KpnI-BssHII enzymes andthe fragment was cloned into the site of the pRC/CMV vector sold by InVitrogen. The final plasmid construction was called pCMV-HBS and wasfiled under No. 1-1370 with the CNCM.

This plasmid is represented schematically in FIG. 1. The CMV promoter issituated between the 288 nucleotide, which is the cleavage position ofMluI, and the 896 nucleotide, which is the cleavage position of KpnI.The DNA fragment including the structural gene of the HBs antigenstructure was cloned between the 896 and 2852 nucleotides (position ofBssH III)

The HBs gene spreads between the 911 (XhoI position) and 2768nucleotides (BglII position), respectively.

The complete sequence for this plasmid is sequence SEQ ID No. 1.

The purified plasmid DNA was prepared by standard methods thenredissolved in PBS buffer and stored at −20° C. until the injection wasperformed.

1.3 DNA Injection

One to five days after the marcaine injection, DNA was injected into thesame area, the mouse being anaesthetized using sodium pentobarbital (75mg/kg interperitonal path).

The DNA solution, which contains 50 μg of plasmid DNA and 50 μl of PBSbuffer, was injected by a single intramuscular injection through theskin into the anterior tibia muscles undergoing regeneration.

The injections were performed bilaterally into the two legs of the mice,each animal thus receiving a total of 100 μg of recombinant plasmid DNA.As for the marcaine injection, the DNA solution was injected using thetuberculosis syringe with the needle described previously.

A single intramuscular DNA injection was performed in each leg.

2. Results

The results obtained are summarized in Table 1 below.

They show very clearly that a DNA injection after treatment withmarcaine allows a large number of seric antibodies to be obtainedagainst the hepatitis B surface antigen.

These results are surprising. From the analysis of the state of the artit was not inferred that a plasmid would allow the induction of anti-HBsantibodies, which could be found in the serum and thus allow aneffective vaccination.

The ease of application of the plasmid vaccination, and the fact thatboosters would not be necessary, allows the consideration of a largescale vaccination.

Example 2 Comparison of the Efficiency of a Plasmid Injection in thePresence and Absence of Lipids

A dose of 10 μg plasmid DNA from the SV40-luciferase vector availablecommercially (“pGL2-Control Vector” from Promega, reference E1 11) in 50μl of physiological solution was injected into the sucrose pretreatedmuscle following the method of Davis et al. (Hum. Gene Ther. 4:151-159(1993)). The injected DNA is mixed earlier with lipids such asdioctadecylamidoglycyl spermine (DOGS) or the following mixtures:DOGS+spermidine, and DOGS+polyethyleneglycol (PEG). The luciferaseactivity was determined 5 days after the injection.

These results are shown in table II below.

They show that the presence of lipids (DOGS) very significantly reducesthe efficiency of the plasmid injection with respect to a compositionwith no lipids (Control).

Example 3 Comparison of the Responses of Mice and Rabbits to PlasmidsCarrying Different Promoters and Envelope Genes for the HBV Virus

Five plasmids were constructed allowing the expression of one, two, orthree envelope proteins for the HBV virus. In three of the constructions(pCMVHB-S, pCMVHB-S2.S, pCMVHB-S1.S2.S) the genes coding for the HBVvirus envelope proteins are put under transcriptional control of thepromoter of the CMV virus precursor genes (FIG. 1, FIG. 2A to 2C, FIGS.3 and 4). The fourth plasmid (pHBV-S1.S2.S) uses the promoter for theHBV virus surface genes contained in the pre-S1 region of this virus(Cattaneo et al. (1983) Nature, 305, 336) (FIG. 2D) as a transcriptionalcontrolling element. In another plasmid, pSVS, the three envelopeproteins were placed under control of the SV40 promoter (pSVS) (Michelet al., Proc. Natl. Acad. Sci. USA, 81:7708-7712 (1984)). Theconstruction of the pSVS plasmid is further described in EP0 156 712 B1.In the five constructions, the polyadenylation signal used is containedin the HBV sequences present in 3′ of the S gene.

1. In Vitro Control of the Vector Efficiency.

To control the efficiency of these vectors in vitro in eukaryotic cells,mouse fibroblasts or myoblasts were transfected. FIG. 6 illustratessecretion kinetics of HBs particles in the culture supernatants. The lowantigen levels produced by transfection of the pCMVHB-S1.S2.S vector arecompatible with a large degree of synthesis of the large envelopeprotein starting from the CMV promoter. This protein being myristilatedin its amino terminal region is retained in the endoplasmic reticulum(Ganem, Current Topics in Microbiology and Immunology, 168:61-83(1991)). Retention in the cell of proteins carrying the pre-S1determinants was confirmed by immunofluorescence.

The composition of the secreted particles was analyzed in an ELISAsandwich system using as capture antibodies a monoclonal mouse antibodyspecific to the pre-S1 (FIG. 7A) or pre-S2 (FIG. 7B) determinants andrabbit anti-HBs polyclonal serum as second antibodies. These experimentsshow that the HBsAg particles produced starting from pCMVHB-S1.S2.Svector carry pre-S1 and pre-S2 determinants showing the presence of thelarge and medium envelope proteins of the HBV virus. Particles secretedafter the transfection of the pCMVHB-S2.S and pHBV-S1.S2.S vectorscarry, in addition to the HBs determinants, pre-S2 determinantscharacteristic of the medium envelope proteins. It was observed that theproportion of pre-S1 and pre-S2 epitopes on the pSVS particles was twicethe amount found on pCMV-S1.S2.S HBsAg particles.

2) DNA Inoculation

DNA purified on a Quiagen column was injected by an intramuscular pathin a single injection of 100 μg (50 μg/leg) in the anterior tibia muscleof mice C57BL/6 (8 mice per group). Five days prior to the injection,the muscle was pretreated with cardiotoxin in order to inducedegeneration followed by regeneration of muscles cells, thus favoringDNA capture by these cells.

The DNA injection experiments were also carried out for rabbits. In thiscase, pCMVHB-S DNA was administered into normal muscle withoutdegeneration, either by using an injection gun without needle calledBiojector®, or by conventional syringes fitted with needles.

3) Anti-Hbs Responses for Mice Vaccinated with DNA

An anti-HBs antibody response is induced by a single injection of anyone of the four plasmids used.

The antibody response was analyzed using a commercial anti-HBs antibodydetection kit (Monolisa anti-HBs, Diagnostic Pasteur). Anti-preS2antibodies are detected by an ELISA system using, on the solid phase, apeptide from the pre-S2 (AA 120-145) region corresponding to a B majorepitope carried by this area (Neuarth et al., 315:154 (1985)).

FIGS. 8A to 8D illustrate the anti-HBs (HBs-Ab) response kineticsexpressed in milli-international units/ml and the anti-pre-S2 response(preS2Ab) determined as optical density (492 nm) for 1/100 dilutedserums. Detection was carried out using a mouse anti-immunoglobulinantibody (IgG) labeled with peroxidase.

The injection of the pCMVHB-S plasmid (FIG. 8A) induces a constantanti-HBs antibody synthesis. Seroconversion was observed in 100% of micefrom one week after the injection with an antibody average level of 48mUI/ml (from 12 to 84 mUI/ml, standard deviation (SD)=28), which is 4 to5 times superior to the threshold required in man to provide protection(10 mUI/ml)

The induced response for a single injection of pCMVHB-S2.S plasmid (FIG.8B) is characterized by the very early appearance of anti-HBsantibodies. These antibodies reach an average level of 439 mUI/ml (from104 to 835 mUI/ml; SD=227) at one week then decline before increasingagain to reach the initial level at 13 weeks. The significance of thisantibody peak will be discussed later. A peak for anti-pre-S2 IgGantibodies is observed at two weeks.

The appearance of anti-HBs antibodies induced by injection ofpCMVHBV-S1.S2.S plasmids (FIG. 8C) and pHBV-S1.S2.S (FIG. 8D) isslightly delayed as the mice only seroconvert to 100% after two weeks.The seroconversion profile is identical; it is characterized by aninitial response, which is specific to the pre-S2 antigen followed by ananti-HBs response, which gradually increases to reach a level of 488mUI/ml (from 91 to 1034 mUI/ml; SD=552) (pCMVHBS1.S2.S) and 1725 mUI/ml(from 143 to 6037 mUI/ml; SD=1808) (pHBV-S1.S2.S) at 13 weeks.

Anti-HBs antibodies were not detected earlier than 2 weeks after pSVSinjection. However, at its peak level (12 weeks), HBs-specificantibodies induced were two times greater than that with pCMV-S1.S2.S.The sera from mice injected with either pSVS or pCMV-S1.S2.S indicatedthat they contained both group-specific (a) and subtype-specific (y)antibodies. However, antibodies in pCMV-S1.S2.S-treated mice appeared toshow a stronger affinity to pre-S2 than those treated with pSVS.

4) Anti-HBS Response of Rabbits Injected with DNA

Results presented in tables III and IV show that the antibody levelsdetected at 8 weeks in rabbits immunized using the Biojector aresignificantly higher than those obtained by a DNA injection by needle.

5) Specificity of Clinically Defined Antibodies

Synthetic peptides were use to determine the relative amounts of pre-S2and pre-S1-specific antibodies for mice injected with pSVS orpCMV-S1.S2.S. The binding to a synthetic peptide pre-S2 peptide(residues 120-145) or pre-S1 peptide (residues 12-49) was measured anddepicted in FIG. 8E. IgM and IgG titers were present after two weeks.While the total titers declined over the next two week, IgG titerscontinued to increase and was maintained for at least 6 months after DNAinjection mice injected with each vector. FIGS. 8F and 8G demonstratethat antibodies to other pre-S2 determinants of the middle protein areinduced after pCMV-S1.S2.S injection, or alternatively, that theresponse to pre-S2 is overwhelmed by anti-pre-S1 antibodies in pSVSinjected mice.

The peptide encompassing residues 12-49 had previously been shown tobind human antibodies specific to pre-S1 on native HBsAg particles(Milich et al., J. Immunol. 137:315-344 (1986)) and peptide 94-117 to bea dominant antibody binding site for murine antibodies (Milich et al.,1986). Antibodies to pre-S1 peptide 12-49, but not to peptide 94-114,were detected in the sera of mice injected with pSVS only. Thespecificity of the antibody induced suggests that the particles producedafter muscle transfection are closely related to particles produced invivo during infection in humans.

FIG. 8H depicts the antibody levels in mice injected with expressionvectors pSVS (squares) and pCMV-S1.S2.S (circles). After two weeks ofDNA injection, 100% of the injected mice had seroconverted to a titer ofat least 10 mIU/ml, which is recognized as a level to be protective inhumans. At 12 weeks, these levels were 50 to 100 times higher.

Example 4 Humoral Responses of Mice to Genetic Vaccine

1) Qualitative Analysis of the Humoral Response

ELISA systems applied to the solid phase of the HBs antigens of varyingcomposition with respect to the determinants presented on the solidphase and using mouse antibodies specific to IgM or IgG as secondantibodies gave a qualitative analysis of the antibody response that wasachieved.

In all cases, the single injection of DNA in mice is characterized bythe early appearance of HBsAg specific to IgM followed immediately byconversion to IgG isotype antibodies, which is characteristic of thememory response induced by the auxiliary T cells. The antibody responseto the DNA injection is characterized by its prematurity. Indeed,seroconversion is achieved 8 to 15 days after the injection depending onthe DNA type used and in all cases the plateau is achieved in four weeksand maintained constantly over a period of 12 weeks.

The use of the heterologous subtype HBs antigens (ad) fixed on ELISAplates allows the formation/detection of the presence, in the serum ofimmunized mice, of antibodies specific to the anti-a group, and bydifference in reactivity with respect to HBsAg of the same subtype (ay),of antibodies specific to the anti-y subtype. The presence of antibodiesspecific to determinants of the HBsAg group is very important as theformer are capable of giving protection against the heterologous subtypevirus during virulent tests in chimpanzees (Szmuness et al., N. Engl. J.Med. 307:1481-1486 (1982)).

Analysis of the response induced by the pCMV-S2.S vector shows that ithas a remarkable similarity with the one, which can be observed in manduring infection. It is characterized by an extremely early (8 days)peak for IgM which is specific to the pre-S2 region immediately followedby conversion to anti-pre-S2 IgG (FIG. 9). This response is followed bythe appearance of IgM then IgG anti-HBs antibodies. The anti-HBsantibody production is constant and reaches a maximum after 4 weeks. At13 weeks IgG anti-HBs and anti-pre-S2 remain at a constant level.

The anti-subtype (y) response precedes that of the anti-group response(a) in the same way as that described for the vaccination with therecombinant vaccine (Tron et al., J. Infect. Dis. 160:199-204).

Thus, injection of a vector encoding the small and the middle forms ofHBV envelope protein (pCMV-S2.S) into normal mice induced a strong andlong-lasting humoral response to the preS2 domain of the middle proteinand to subtype- and group-specific HBsAg determinants.

The response obtained with the three other DNA vaccines illustrates thecommutation of class IgM-IgG, which is characteristic of the secondaryresponse.

The response being first of all directed against the subtype beforebeing against the HBsAg group determinants.

The long term response, which was studied for pCMVHB-S DNA, shows thatthe antibody peak is reached within 3 months and this remains at aconstant level 6 months later (Table V) (Davis et al., Vaccine, 14(9):910-915 (1996)).

2. Immunization of Mice with Genetic Vaccine and Non-Response

The high number of non-responders to the classical vaccine (2.5 to 5%)remains a major problem for vaccination against hepatitis B. It has beenpossible to correlate the non-response in man to certain HLA types(Krustall et al., J. Exp. Med. 175: 495-502 (1992)) and to a defect inthe antigen presentation or stimulation of the auxiliary T cells.

To study the possible impact of the genetic vaccination on the HBsAgnon-response, a range of mice strains that were used for which theresponse to various HBV virus envelope proteins is controlledgenetically and has been well characterized by Millich et al. (J.Immunol. 137:315 (1986)). The pCMVHB-S construction previously describedwas injected into B10 (H-2^(f)), B10.S (H-2^(s)), and B10.M (H-2^(f))mice muscles.

The B10 strain responds to the three virus envelope proteins. The B10.Sstrain does not respond to HBsAg, however this non-response can beovercome by immunization with HBsAg antigens which are carrying pre-S2determinants. The B10M strain is totally non-responsive to both HBs andpre-S2 antigens. A response for the latter strain can be achieved byimmunization using HBsAg carrying pre-S1 determinants.

The mice immunized by the DNA received a single injection (100 μg) inthe regenerating muscle. Control mice were injected with twointraperitoneal injections of protein at an interval of one month, thefirst of 2 μg HBsAg to which the complete Freund additive (CFA) wasadded and the second of 2 μg HBsAg to which the incomplete Freundadditive (IFA) was added.

The results obtained for pCMVHB-S are illustrated by FIGS. 10A to 10F.

In the B10 strain (good responder), the DNA-induced response is earlierthan that induced by the protein after a single injection.

The appearance of anti-HBs antibodies subtype specific then groupspecific after immunization with pCMVHB-S DNA was observed in the B10Sstrain (nonresponder to HBsAg in the absence of pre-S2). Group specificanti-HBs antibodies are observed in HBs protein immunized mice onlyafter the second injection.

A group and subtype specific anti-HBs response is obtained for DNAimmunization of strain B10M (nonresponder to HBsAg in absence ofpre-S1), whereas only a subtype specific response is induced by theprotein with two injections being required.

The response induced by the three vector types is compared in the threemice strains.

3. Genetic Vaccine in HBsAg Transgenic Mice

Transgenic mice of the C57BL/6 strain is used as a model of chronic HBVcarriers since they constitutively express HBsAg. A single injection ofthe pCMV-S2.S DNA into HBsAg-transgenic mice (H-2^(b)) provoked adecrease in titres of circulating HBsAg (FIG. 12) and the concomitantappearance of anti-HBs antibodies, which increased over time (FIG. 13).In some of the mice, antigen was eliminated from the serum as early asfour weeks after injection of the DNA and remained undetectable for atleast twelve weeks without further injections of DNA. In the remainingmice, antigen levels also fell and were maintained at low levels. Theseeffects were not due to non-specific immune stimulation induced by theinjection procedure or the presence of DNA per se, since HBsAg levelswere unaffected (FIG. 12) and no anti-HBs were detected (FIG. 13) incontrol transgenic mice injected with PBS alone or a DNA vectorexpressing beta-galactosidase (β-gal; pCMV-LacZ) (Davis, 1993), eventhough the latter procedure induced high levels of anti-B-gal antibodies(ELISA titres>10⁵ by 12 weeks post immunization).

Free antibodies were first detectable in the plasma of transgenic mice2-4 weeks following a single injection of pCMV-S2.S DNA (FIG. 13). Thefirst antibodies detected were preS2-specific since they reacted onlywith particles carrying this epitope but not with particles devoid ofit. Anti-HBs antibodies were not observed until 8 weeks, at which timethere was a complete clearance of circulating HBsAg (see FIG. 12). It isremarkable that, even though the transgenic mice had been tolerant tohigh levels of circulating HBsAg, DNA-based immunization was able toinduce titres of anti-HBs comparable to those induced in non-transgeniccontrols, and that these antibodies were able to completely neutralizethe circulating HBsAg. The isotype profile of the anti-HBs antibodieswas identical in transgenic and in non-transgenic mice and included IgG2as well as IgG1 with some IgG3 (FIG. 14). Such isotype switching ofautoreactive B cells strongly suggests that the DNA-mediatedimmunization triggered CD4+ T helper cells.

3.a. Cytokine Production from Spleen Cells of Transgenic andNon-Transgenic Mice

To further characterize the T-helper subset, the cytokine productionfrom spleen cells in culture was analyzed. Spleens were removed fromtransgenic mice 20 weeks after DNA injection and cell suspensions werespecifically stimulated in vitro with HBsAg. These cultures producedγ-interferon (IFN-γ), low levels of IL-2 and TNF-α, but no IL-4 asdepicted in Table VII. The secretion of IgG2a and γ-interferon isconsistent with a predominant Th1 response, however, detection of IgG1suggests that the Th2 response was also induced.

3.b. Regulation of Transgene Expression in pCMV-S2.S VaccinatedTransgenic Mice

The rapid clearance of circulating antigen from immunized mice did notappear to result from a significant or persistent HBsAg-specificcytopathic effect on the liver since levels of transaminase activity inthe plasma remained normal subsequent to injection of DNA andhistological examination of the liver at the time of HBsAg clearanceshowed not evidence of necrosis or inflammation (not shown).

Since the inventors could not correlate the persistent clearance of thetransgene product with any apparent destruction of thetransgene-expressing liver cells, the HBV mRNA content in the livers oftransgenic B10 mice was evaluated. At 12 weeks after immunization withpCMB-S2.S, the mRNA was decreased in the livers of those mice which hadpartially cleared the antigen, and was undetectable in those which hadcompletely eliminated HBsAg from their sera (FIG. 15A, lanes 5 and 6-7respectively). This effect is persistent since HBV mRNA remainedundetectable in the livers of mice analyzed 20 weeks after DNA injection(FIG. 15A, lanes 8-9). In contrast, HBV mRNA was not diminished inlivers taken from untreated transgenic mice or control transgenic mice,which had been injected with pCMV-LacZ DNA (FIG. 15A, lane 1 and lanes2-4 respectively). This indicates that the inhibition of viral geneexpression in transgenic mice injected with pCMV-S2.S was not due to anon-specific effect such as the release of cytokines withinjection-induced inflammation and/or with an immune response againsttransfected muscle cells expressing a foreign antigen (i.e., β-gal).Thus, the HBsAg-specific immune response induced by immunization withplasmid DNA appears to be responsible for controlling hepatic transgeneexpression by some non-cytopathic mechanism.

3.c. Injection of DNA Induced B and T-Cell Response to HbsAg

To determine which component of the immune response is implicated in thedown-regulation of HBV-specific mRNA and in the observed decrease orelimination of the circulating antigen, adoptive transfer experimentswere performed. Fully immunocompetent non-transgenic mice were immunizedonce with the pCMV-S2.S DNA vector and when ELISA titres of serumantibodies to HBsAg had reached at least 10⁴, both the serum and theprimed spleen cells were harvested from the mice for transfer into theirtransgenic littermates.

Passive transfer of serum-derived antibodies on a single occasion intotransgenic mice induced a rapid but transient decrease in circulatingHBsAg levels (mice 2.21 and 4.26, Table VI). In other transgenic mice,circulating antigen was maintained at undetectable levels for a longerperiod by intra-peritoneal injection of hyperimmune sera every 2-3 daysover a period of 17 days (mice 1.3.5 and 1.3.6, Table 2). Neither singlenor chronic administration of antibodies resulted in decreasedHBV-specific mRNA in the liver (not shown) indicating that the humoralresponse after DNA-based immunization was not responsible for thedown-regulation of transgene expression or the long-term elimination ofthe transgene product.

Injection of primed spleen cell suspensions obtained from pCMV-S2.Simmunized non-transgenic donor mice into transgenic littermaterecipients resulted in a rapid clearance of circulating HBsAg (by 7days) and a concomitant appearance of anti-HBs antibodies, which weresustained (FIG. 16). This indicates that the transferred T and B cellswere functional within the environment of the transgenic mice and thatthe B cells were activated, probably by the circulating antigen.Adoptive transfer of HBsAg-primed spleen cells was also able to induce acomplete disappearance of HBV mRNA in the liver by 17 days (FIG. 17 andFIG. 15B, lanes 4, 5). The inability to detect serum HBsAg occurred 10days prior to the disappearance of HBV mRNA suggesting that two separatemechanisms may be responsible for the observed clearance of the antigen:an initial transient elimination due to formation of immune complexesand a subsequent more permanent control of transgene transcription.Serum transaminase activity and histological examination of liversections were monitored every 2-3 days after adoptive transfer and foundto be normal for up to 17 days, after which some histological sectionsof liver exhibited a few small necrotic foci. These were sometimesaccompanied by the presence of inflammatory cells, but in no case didthe necrotic regions involve more that 5% of the hepatic cells. Inaddition, a few apoptotic hepatocytes were detected in centrilobularareas within some randomly distributed lobules (not shown). The changesinduced by adoptive transfer with HBsAg-primed spleen cells wereHBsAg-specific since injection of β-gal-primed spleen cells intotransgenic recipients had no effect on the levels of serum HBsAg (FIG.16) or liver HBV mRNA (FIG. 14B, lanes 2, 3).

3.d. T Cells are Able to Control Transgene Expression in the Absence ofAntibody Production.

To determine the spleen cell population, which was involved in thedecrease or disappearance of circulating HBsAg and liver HBV mRNA,adoptive transfer was carried out with fractionated B- or T-spleen cellsobtained from non-transgenic donor mice immunized with pCMV-S2.S. Afterdepletion of T-cells, the transfer of HBsAg-primed spleen cells intotransgenic mice did not induce anti-HBs and had no effect on levels ofcirculating HBsAg or liver HBV mRNA (FIG. 15B, lane 6). This indicatesthat antibody production in DNA-immunized transgenic mice is T-celldependent. In contrast, transfer of B cell-depleted spleen cellsresulted in clearance of circulating HBsAg within 14-17 days althoughantibody to HBsAg was not detected in these recipient mice at these orlater times (not shown). Thus, antigen-antibody complex formation is notrequired for, but has a synergistic effect on the elimination ofcirculating antigen (FIG. 16). Furthermore, it appears thatHBsAg-specific T cells are also responsible for down regulation oftransgene expression since only transfer of T—but not B—or unprimedT-cells was able to reduce HBV mRNA in the liver to undetectable levels(FIG. 15B, lane 6-9).

4. Genetic Vaccine in Clinical Trials

H. A. Thoma, Progress in Hepatitis B Immunization, P. Coursaget and M.J. Thong (Eds.), Colloque INSERM: Paris, France, 194:35-42 (May 3-5,1990), the entire contents of which are incorporated by referenceherein, reports that a third generation vaccine containing portions ofpre-S1, pre-S2, and S proteins demonstrates fast and high immuneresponse in clinical trials in man as compared to other availablevaccines.

It is generally thought that the humoral response to HBs antigens issufficient by itself to give protection. The presence of antibodiesdirected against other determinants (pre-S1 and pre-S2) carried by thevirus envelope proteins, themselves protectors, could improve theresponse quality. The experiments reported here as a whole illustratesthat the humoral response induced by the genetic anti-hepatitis Bvaccination is greater in several fields than that which can be achievedfor the classical vaccination.

In terms of seroconversion levels, the 100% level is obtained, afteronly one injection, from day 8 for mice immunized with pCMV-HBS DNA andpCMVHB-S2.S.

In terms of response level, the 10 mUI/ml threshold level, consideredsufficient to give protection in man, is always greatly exceeded.

In terms of the speed of response, in 8 days a very high level ofanti-pre-S2 antibodies is obtained for the pCMVHB-S2.S vector and it isknown that the former are capable of giving protection by themselves(Itoh et al., (1986) Proc. Natl. Acad. Sci. USA 83, 9174-9178).

In terms of response stability, anti-HBs antibodies remain constant at ahigh level for more than 6 months.

In terms of response quality, type IgG antibodies characteristic of aresponse, which is dependent on the auxiliary T cells and therefore on amemory response, are obtained. Moreover, the single injection of DNAencoding the HBV protein is sufficient to break T-cell tolerance intransgenic mice expressing the same envelope sequences in the liver.

In terms of anti-viral activity, the antibodies are specific to theviral subtype, but especially group-specific and therefore susceptibleto giving a cross protection and to clear HBsAg particles in HBsAgtransgenic mice.

In terms of biological significance, the response profile obtained bypCMVHB-S2.S immunization mimes totally that which is observed in manafter a resolved viral infection.

The immune response resulting from plasmid expression of the HBsAg leadsto both clearance of circulating antigen as well as the induction ofT-cell responses capable of suppressing HBV mRNA accumulation in theliver in a transgenic mouse model. Despite the high concentration oftransgene product in the circulation, auto-antibodies are induced soonafter DNA injection, although initially these are directed only againstthe preS2 epitope.

In terms of treating HBV chronic carriers, the murine model shown heredemonstrates that T-cell non-responsiveness can be overcome by usingDNA-mediated immunization. The induced response mimics in some aspectsthat required to clear a viral infection, namely an adequate cellularimmune response to regulate viral gene expression without killinginfected cells and an adequate humoral response to prevent the spread offree virus to uninfected cells. Thus, the inventors have arrived at thefirst demonstration of an immunotherapeutic application of DNA-mediatedimmunization against an infectious disease and particularly, provides atreatment for HBV chronic carriers.

TABLE I Induction of antibodies against the hepatitis B surface antigenLevel of antibodies against hepatitis B surface antigen in Number of theserum (mIU/ml) 15 days after 35 days after Description mice Before DNAinjection DNA injection DNA injection DNA injected 1 day 5 0 average: 56from average: 59 after marcaine 5 to >140 treatment DNA injected 5 days5 0 average: 71 from average: 47 after marcaine 21 to >100 treatment

TABLE II Luciferase RLU/sec/muscle (Average ± SEM) RLU = Percentagerelative Group Relative Light Unit to the control Control 43 082 ± 5 419 100% 4X DOGS 20 ± 7 0.06% DOGS - Spermidine  50 ± 23 0.12% PEG-DOGS  0± 0 0.00%

TABLE III Immunization with the Biojector^(R) pCMV-HB.S N° 0 weeks 2weeks 8 weeks 2.1 0 517 380 2.2 0 374 322 3.1 0 250 418 4.1 0 400 40454.2 0 88 86 4.3 0 314 420 6.1 0 415 1001 6.2 0 1543 3517 6.3 0 1181 141Average 0 566 mUI/ml 1148 mUI/ml SD 0 476 1521 SEM 0 159 507 N 9 9 9 CV84% 133%

TABLE IV Immunization by injection using a needle pCMV-HB.S N° 0 weeks 2weeks 8 weeks 1.1 1 0 1 5.1 0 287 186 5.2 0 162 798 5.3 0 305 203 7.1 086 175 7.2 0 1100 dead Average 0 325 mUI/ml 273 mUI/ml SD 0 401 305 SEM0 164 136 N 6 6 5 CV 245% 124% 112%

TABLE V Long term response of a mouse vaccinated with pCMVHB-S 1 2 3 6month months months months * a-HBs titre 227 662 1299 1082 in mUI/ml *a-HBs 3.5 × 10⁻⁴ 5 × 10⁻⁴ 0.5 × 10⁻⁴ 9 × 10⁻⁴ ELISA titre

Table VI

Serum titres of HBsAg in Tg mice passively transferred with antibodiesto HBsAg. Results are shown for seven Tg mice injected intraperitonealyonce (mice 4-23, 2-21, 6-11 and 4-26) or every 2-3 days (mice 1-3-16,1-3-5, 1-3-6) with either anti-HBs immune sera (anti-HBs Ab) or normalmouse sera (NMS). HBsAg titers (ng/ml) were determined in the seracollected at the indicated time. Mice were killed (†) 26 hours or 17clays after the transfer and their livers were harvested for extractionof mRNA and Northern blot analysis. ND: Not Done

Injected Bleeding (days) Mouse n^(o) serum 0 0.25 1 2 3 6 10 15 17 4-23NMS 429 404 477 420 440 452 ND 252 502 † 2-21 anti-HBs Ab 1321 0 13 61373 725 ND 1028 1356 † 6-11 NMS 696 548 442 † 4-26 anti-HBs Ab 1080 0 22† 1-3-16 NMS 565 ND 542 ND 326 562 328 647 693 † 1-3-5 anti-HBs Ab 721ND 0 ND 0 0 0 3 0 † 1-3-6 anti-HBS Ab 548 ND 3 ND 0 0 3 6 0 †

Table VII

Secretion of cytokines by spleen cells in culture. Splenocytes ofpCMV-S2.S-immunized Tg and non-Tg mice were incubated with medium orstimulated with concanavalin A (ConA, 2.5 μg/ml), preS2 peptide (10μg/ml) or HBsAg particles (3 μg/ml) for 72 hr. Antigen-specific culturesupernatants were harvested for determination of cytokine levels (pg/ml)at 24 hr for TNF-α and IL-2 determinations and at 48 hr for IFN-γ andIL-4. Data are as the arithmetic mean±SEM of 5 to 6 spleensindependently tested in two experiments.

Mice Cytokines Medium ConA preS2 peptide HBsAg Non-Tg IFN-γ 2 ± 1 1,611± 377  92 ± 42 60 ± 28 TNF-α 28 ± 13 846 ± 87 267 ± 23  58 ± 17 IL-2 2 ±1 2,584 ± 233  3 ± 2 4 ± 2 IL-4 6 ± 4  62 ± 15 4 ± 4 8 ± 6 Tg IFN-γ 2 ±2 1,709 ± 12   329 ± 227 39 ± 25 TNF-α 28 ± 12 871 ± 13 405 ± 137 83 ±46 IL-2 1 ± 1 4,136 ± 578  1 ± 1 1 ± 1 IL-4 11 ± 8  49 ± 3 12 ± 6  11 ±9 

1. A nucleotide sequence comprising a promoter homologous to the hostand another regulatory sequence for the expression of a gene or a DNAcoding complement for S, preS₂-S, or preS₁-preS₂-S.
 2. A vaccinecomprising the nucleotide sequence according to claim
 1. 3. Acomposition capable of inducing a cytotoxic response comprising a geneor complementary DNA coding for at least a portion of a virus protein,which is expressed in the muscle cells, and an internal promoter.
 4. Thecomposition of claim 3, wherein said gene is the S gene.
 5. Thecomposition of claim 3, wherein said protein is the S, preS₂-S, orpreS₁-preS₂-S protein.
 6. A non-lipid pharmaceutical compositioncomprising at least one substance capable of inducing a coagulatingnecrosis of the muscle fibers and either a vector comprising a gene orcomplementary DNA coding for at least a portion of a virus protein, anda promoter allowing the expression of the gene in the muscle cells or acomplete or partial nucleotide sequence comprising a promoter homologousto the host and another regulatory sequence for the expression of a geneor a DNA coding complement for S, preS₂-S, or preS₁-preS₂-S.
 7. Thecomposition of claim 6, wherein said gene is the S gene.
 8. Thecomposition of claim 6, wherein said protein is the S, S-preS₂, orS-preS₁-preS₂ protein.
 9. The composition of any one of claims 6 to 8,wherein said composition is administered to a chronic HBV carrier.
 10. Acomposition for inducing a B and/or T cell response in chronic HBVcarriers comprising a vector, wherein said vector comprises a gene orcomplementary DNA coding for at least a portion of a hepatitis B proteinand a promoter.
 11. The composition of claim 10, wherein the B cellresponse is able to clear the circulating HBsAg.
 12. The composition ofclaim 10, wherein said gene or complementary DNA encodes a HBV proteinselected from group consisting of pre-S2, pre-S1, S protein, and a partof pre-S2, pre-S1, or S protein.
 13. The composition of claim 10,wherein said gene or complementary DNA encodes a pre-S2 or pre-S1protein of HBV and an S protein of HBV or a part of pre-S2 or pre-S1protein and a part of S protein.
 14. A composition according to any oneof claims 10, 11, 12, or 13 for inducing a T cell response.
 15. Aprocess of treating chronic HBV carriers comprising administering tosaid chronic HBV carriers a therapeutically effective amount of thecomposition according to any one of claims 10 to
 13. 16. A recombinantplasmid containing a nucleotide sequence according to any one of claims10 to 13, wherein said sequence encodes for the small and/or the middleforms of HBV surface protein.